Although protein phosphatase activity in the cell appears to change in response to hormonal signals (review: Curnow, R.T. and Larner, J., Biochem. Act. of Hormones, VI, Chapt. 2, pp. 77-119, Academic Press (1979), nothing is known of the molecular basis for these hormonal effects. This is largely because purified native phosphatases have not been available for these studies. In the proposed project, native phosphatases will be purified and their regulatory properties studied. The native forms of phosphorylase phosphatase and glycogen synthase phosphatase will be purified from rabbit skeletal muscle. Special precautions will be taken to minimize the possibility of artifact generation during the purification process. An alternative approach to studying native phosphorylase phosphatase will involve attempts to use Sepharose-bound phosphorylase phosphatase catalytic subunit to isolate potential phosphatase regulator proteins in muscle extracts. Proteins which bind to the phosphatase affinity column will be assayed for their ability to inhibit phosphorylase phosphatase catalytic subunit and to combine with the catalytic subunit to reconstitute the native enzyme. The effects of various metabolites including cyclic-AMP, cyclic-GMP, and calcium ion on the purified native phosphatases will be investigated. Studies will be initiated to determine whether the purified phosphatases or regulatory proteins can be phosphorylated by various protein kinases, and whether the phosphorylation affects the activity of the phosphatases. It has been demonstrated in several studies that synthase phosphatase, phosphorylase phosphatase and glycogen synthase appear to have different properties in diabetic rats. Studies will be initiated to determine whether these enzymes are modified in vivo by calcium-dependent proteases in skeletal muscle extracts from starved and diabetic rats.